Gallyas silver staining

For silver staining, paraffin-embedded tissue on slides were rehydrated in xylene and series of ethanol solutions (100%, 90%, and 70%) followed by a washes in dH₂O. Sections were incubated in 5% periodic acid for 5 min, washed twice in dH₂O for 5 min and placed in alkaline silver iodide (4% sodium hydroxide, 10% potassium iodide and 0.035% silver nitrate in 100 mL dH₂O) for 1 min. Following a 10 min wash step in 0.5% acetic acid, the sections were placed in developer solution for 5–10 min until desired staining was achieved (Stock solution I: 5% sodium carbonate in water; Stock solution II: 0.2% ammonium nitrate, 0.2% silver nitrate, 1% tungstosilicic acid in water; Stock solution III: 0.2% ammonium nitrate, 0.2% silver nitrate, 1% tungstosilicic acid, 0.73% formaldehyde in water; 3 volumes of stock solution II added to 10 volumes of stock solution I followed by 7 volumes of stock solution III). Subsequently, sections were washed in 0.5% acetic acid for 3 min, rinsed in dH₂O for 5 min and incubated in 0.1% gold chloride for 5 min. After a 5-min rinse in dH₂O, sections were incubated in 1% sodium thiosulphate for 5 min and rinsed in tap water. Sections were counterstained with nuclear fast red (Thermo Fisher Scientific, Waltham, MA) according to manufacturer’s instructions. Slides were dehydrated in ethanol solutions (70%, 90%, and 100%) and xylene before they were covered with Cytoseal (Thermo Fisher Scientific, Waltham, MA). Slides were digitally scanned using an Aperio Slide Scanner AT2 instrument (40X magnification; Aperio Technologies Inc., Vista, CA).